Strontium isotope analysis of otoliths reveals differences in the habitat salinity among three sympatric stickleback species of the genus Pungitius

The analysis of otolith Sr isotope ratios (87Sr/86Sr) is a powerful method to study fish migration in freshwater areas. However, few studies have applied this method to study fish movement in brackish‐water environments. Furthermore, despite the fact that habitat differentiation has been shown to drive genetic differentiation and reproductive isolation among stickleback fish, no studies have used the otolith 87Sr/86Sr ratios to analyze habitat differentiation between stickleback ecotypes and species. In this study, we analyzed the otolith 87Sr/86Sr ratios of three sympatric stickleback species of the genus Pungitius in the Shiomi River on Hokkaido Island, Japan: P. tymensis, the brackish‐water type of the P. pungitius–P. sinensis complex, and the freshwater type of the P. pungitius–P. sinensis complex. First, we created a mixing equation to depict the relationship between habitat salinity and the 87Sr/86Sr ratios of river water. We found that the otolith 87Sr/86Sr ratios differed significantly among the three species, indicating that the three species utilize habitats with different salinities: P. tymensis and the brackish‐water type inhabit freshwater and brackish‐water environments, respectively, with the freshwater type using intermediate habitats. In addition, we found that some freshwater individuals moved to habitats with higher salinities as they grew. Our study demonstrates that the analysis of otolith 87Sr/86Sr ratios is a useful method for studying the habitat use of fish in brackish‐water environments and habitat differentiation among closely related sympatric and parapatric species. We analyzed the otolith Sr isotope ratios of three sympatric stickleback species of the genus Pungitius in the Shiomi River on Hokkaido Island, Japan. We created a mixing equation to depict the relationship between habitat salinity and the Sr isotope ratios of river water. We found that the otolith Sr isotope ratios differed significantly among the three species, indicating that the three species utilize habitats with different salinities.